Northern Blot
1. Dry down 10-20μg total RNA (or 1-2μg polyA+ RNA) in the speed vac. and dissolve in
10-12μl-sample buffer.
2. Heat at 65℃ for 10-15 min and transfer samples to ice.
3. Add 1μl Ethidium Bromide (1mg/ml), mix and load gel (1X MOPS/EDTA Running buffer).
4. Run the gel at 30V for 20 hours or 100V for 3-4 hours.
(if run fast it is necessary to have circulation of running buffer).
After electrophoresis the RNA can be visualized under UV. (16S RNA should be ¾ down).Take picture with ruler.
5. Treat gel for 30 min in 50mM NaOH, 150mM NaCl.
Then 30 min in 0.1M Tris pH7.5, 150mM NaCl.
Transfer to Membrane
Membrane (Biodyne®B 0.45 u, Pall Corporation) is cut to fit and soaked in H2O and 20X SSC
Build sandwich as shown below (red is weight).
Paper towels
Whatman paper
Membrane
Gel
Isolation of transfer to gel area
Whatman paper
Gel tray or 20X SSC soaked sponge
The glass chamber is filled with 20X SSC, the first piece of whatman paper (from the bottom) has
to touch the buffer on both sides of the sponge or gel tray.
Leave this construct overnight for transfer of RNA from gel to membrane
Next morning, UV autocrosslink, then bake at 80℃, 30min
Probe labeling
RadPrime DNA labelling System (Invitrogen cat no 18428-011)
1. Denature 25ng DNA dissolved in 5-20μl of sterile distilled water or TE in a microcentrifuge tube by heating for 5 min in boiling water bath, immediately cool on ice.
2. Add 3μl of dNTP mix (500μM each dATP, dGTP, dTTP)
3. 20μl 2.5X Random Primers Solution
4. 5 μl (approx 50 μCi [α-32P]dCTP, 3000 Ci/mmol, 10mCi/ml
5. Distilled water to a total volume of 49 μl
6. Mix briefly
7. Add 1 μl Klenow Fragment
8. Incubate at 37℃ for 30 min.
9. Purify the probe using “PCR purification kit” or “Nucleotide removal kit” from Qiagen
10. Add 500μl PN buffer to the sample and transfer sample to spin columns. Spin for 1 min at 14000rpm, discard flow-through as radioactive waste
11. Wash with 600μl PE buffer, spin 1 min, 14000 rpm, repeat wash
12. Discard waste and spin 1min to spin columns completely dry (1min, 14000 rpm).
13. Elute samples by adding 200 μl TE and spin into a clean eppendorf tube (1min, 14000 rpm).
